The invention relates generally to methods and compositions for conducting diagnostic testing on preserved biological samples, and, in particular to performing nucleic acid extraction and amplification methods on such preserved samples.
In the fields of medical diagnosis and medical research, samples (e.g., tissues) are taken from a patient or subject (e.g. a human patient, a human subject, an experimental animal model of a human disease) to determine the condition of the subject in support of the research, determine the current condition of the patient for making a medical diagnosis, determine the response of the patient to a current course of therapy or treatment, etc.
Samples that are obtained for analysis, either for performing medical diagnosis or for use in scientific research, are often placed in a special transport/preservative medium to keep the sample from degrading or decomposing when removed from the subject. Thus, the sample will as closely as possible be in the exact condition it was in when removed from the subject. This ensures that the sample accurately reflects the state of the patient or subject at the time of sampling and will therefore yield the most accurate result in any subsequent studies of the sample. Some of these studies will involve nucleic acid (for example deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)) extraction and amplification.
Extracting DNA and/or RNA from biological samples requires, among other steps, lysis of the cell wall (in the case of prokaryotic cells), lysis of the cell membrane (in the case of certain eukaryotic cells) or lysis of the viral capsid (in the case of viruses). Subsequent amplification requires, in part, the attachment of primers to specific sites within the target nucleic acid.
There are a number of protocols available for the extraction and subsequent amplification of nucleic acid. Some of these protocols utilize high throughput devices. High throughput devices are automatic in the sense that a sample is placed within the device together with appropriate chemicals and the extraction and amplification steps take place without further input from the operator (e.g. Viper™ XTR System by Becton Dickinson® BD ProbeTec Qx Amplified DNA Assay Package Insert, Becton Dickinson 2008). Other protocols utilize less sophisticated equipment and are typically referred to as manual procedures. However, regardless of the protocol, the need to lyse the cells or viral capsids in order to release the nucleic acid, the need to attach the nucleic acid to particles such as ferric oxide in order to extract the nucleic acid from the rest of the sample, and the need to attach primers to the target nucleic acid in order to amplify the nucleic acid remain.
Transport media (e.g. liquid cytology media) typically contains one or more constituents that preserve certain cells in one or more ways (e.g., prevent the breakdown of the cell wall or the cell membrane by cell lysis). In addition, some of these constituents serve the dual purpose of preservation and decontamination of the sample. These constituents are known to interfere with the ability to lyse cell membranes and walls, the ability for the extracted nucleic acids to attach to particles utilized in nucleic acid extraction, and the ability to amplify target nucleic acid.
Liquid based cytology compositions such as SurePath® (Tripath Imaging, Inc., N.C.) solution or ThinPrep® PreservCyt® solution (Hologic Inc., MA), adversely affect the ability to extract amplifiable target nucleic acid from samples exposed thereto. Many reasons have been suggested to explain this observed adverse effect including: 1) degradation of the nucleic acids by constituents in the media; 2) chemical alteration of the nucleic acids by constituents in the media; and 3) inhibition of the cell lysing mechanism in the tissue, which inhibits the release of the nucleic acids for extraction and amplification. In order to avoid the adverse effects of the liquid cytology compositions on extracted DNA, cells are extracted from the compositions prior to lysis. Typically, the extraction requires centrifugation to decant the liquid cytology composition from the cells. The cells are then resuspended in a buffer and lysed with an enzyme. Such extra steps are not typically compatible with many high throughput automatic devices such as, for example, the aforementioned Viper™ System. Even in situations where automation is not involved, such steps are nevertheless time-consuming. The additional time required by these steps can delay obtaining the test results and is preferably avoided.
One example of a media kit for purification of nucleic acids is the QIAamp MinElute Media Kit from Qiagen. This media kit is described in the QIAmp MinElute Media Handbook dated February, 2004. The QIAamp procedure is described as having 4 steps: lyse, bind, wash and elute. In this procedure, the samples are lysed using proteinase K followed by binding the nucleic acids to the QIAmp MinElute column by absorption onto the silica-gel lysate. Although the QIAamp procedure is a proven method, it is optimized for the purification of only 250 μl of liquid cytology media and is both labor intensive and time consuming (i.e. it requires 18 steps that includes 65 minutes of different temperature incubations, 5 centrifugation steps, 2 vacuum filtration steps and several mixing steps). The use of such multi-step methods has heretofore been required to successfully purify nucleic acids from fixed samples such as liquid cytology media and paraffin embedded tissue. It is well known that purification of nucleic acids from fixed media is more difficult than from fresh tissue because the fixatives in the media introduce undesirable chemical modifications of the target molecules in the sample. The reactions can occur to crosslink or otherwise modify nucleic acids in a sample. Other additives in the transport media can also cause undesired cross-linking. For example, cross-linking due to the presence of formalin in the transport media is described in Rai, V. K., et al., “Modeling formalin fixation and antigen retrieval with bovine pancreatic ribonuclease A:I-Structural and functional alterations,” Lab. Invest. Vol. 84(3):292-299 (March 2004). Formaldehyde also produces cross-linking between nucleic acids and proteins as described in Solomon, M. J., “Formaldehyde-mediated DNA-protein crosslinking: A probe for in vivo chromatin structures,” Proc. Natl. Acad. Sci. Vol. 82, pp. 6470-6474 (October 1983). As described in Sepp, R., et al. “Rapid techniques for DNA extraction from routinely processed archival tissue for use in PCR,” J. Clin. Pathol. Vol. 47:318-323 (1994), DNA extraction from cells taken from formalin-fixed paraffin wax typically requires processing steps (e.g. prolonged boiling) that can adversely affect the amount of DNA for amplification. According to Sepp, R., et al. boiling the sample is needed to, among other things, inactivate the proteinase K. Furthermore, proteinase K is inhibited by the constituents in many fixatives making its direct use of limited effectiveness in breaking down protein cross-links.
Therefore, methods and compositions for extracting DNA from tissues and other cells and cell components that overcome the problems of cross-linking and other undesired modifications to nucleic acid yet do not require prolonged high temperature processing that can adversely affect the sample or make the process more expensive and time consuming continue to be sought.